Title: ?Control of DNA Topology? PI: Yuk-Ching Tse-Dinh, PhD Project Summary/Abstract The long term goals of this project are to understand how the activity, regulation and interactions of DNA topoisomerases control DNA topology and affect vital cellular functions. Drugs that target type IB and type IIA topoisomerases are used in current anti-cancer and anti-bacterial therapy. Bacterial type IA topoisomerases have been validated as a useful target for discovery and development of novel antibacterial therapy to treat drug resistant bacterial pathogens that cannot be eliminated with current antibiotics, including the antibiotics that target bacterial topoisomerase II. The proposed research activities for the next funding period would elucidate the complete catalytic mechanism and provide new insights into the protein-protein interactions and regulation of Escherichia coli topoisomerase I activity. This information is needed to fully realize the potential of discovery of novel antibacterial drugs specific for type IA topoisomerases that is present in every bacterial pathogen as a potential therapeutic target. Topoisomerase I catalyzes the relaxation of negatively supercoiled DNA by cleaving a single DNA strand in the underwound duplex DNA and passing the complementary DNA single strand through the break before religation of the cleaved strand to increase the DNA winding. The molecular mechanism of the large enzyme conformational changes that are required for the coordinated movement of the passing DNA is the critical barrier for elucidating how bacterial topoisomerase I can relax negatively supercoiled DNA with high efficiency to prevent hypernegative DNA supercoiling and R-loop stabilization that can arise during transcription elongation. Structural and biochemical studies will be conducted to test hypotheses generated from crystal structures obtained in this project on the mechanism of enzyme DNA conformational change and DNA passage. Interaction sites of endogenous bacterial toxins that can act as inhibitors of topoisomerase I catalytic activity will be identified. Our preliminary results showed that deacetylation of topoisomerase I may be an important function of the E. coli deacetylase CobB. Novel mechanisms of regulation of DNA topology and bacteria growth via acetylation-deacetylation of topoisomerase I will be investigated. The project will include direct participations of co-Investigators who are experts in structural biology and single molecule studies of enzyme-DNA interactions. The results will provide the molecular basis of bacterial topoisomerase I function, and new insights into growth regulation of bacteria via modulation of topoisomerase I enzyme activity. Success in these experiments would support the drug discovery efforts of utilizing bacterial topoisomerase I as a new target for antibiotics.